The present invention relates to novel quenching reagents and to methods for their use in the purification of synthetic intermediates and products in the practice of organic synthesis, combinatorial chemistry, and automated organic synthesis.
Combinatorial chemistry and automated organic synthesis have proven to be highly effective means for the generation of multiplicities of novel molecules known as libraries. As the size of such a library grows, so does the likelihood that it will contain individual molecules with useful biological activities which may be employed in the treatment of human, animal, and plant diseases. Research organizations that can prepare and screen a large number of diverse compounds efficiently, have an increased likelihood of discovering and optimizing new products. For recent reviews in the use of combinatorial chemistry in pharmaceutical discovery see Gallop M. A., et al., J. Med. Chem., 1994;37:1233, Gordon E. M., et al., ibid., 1994;37: 1385, Terret N. K., et al., Tetrahedron, 1995;51:8135, and Ellman J. A., et al., Chem. Rev., 1996;96:555.
In the practice of organic synthesis, the most time consuming element is typically the purification of the desired product following each synthetic transformation. Traditionally, automated organic synthesis and combinatorial chemistry have relied on a number of methods to reduce the amount of time and effort devoted to purification. Such methods include water soluble reagents, polymer-supported reagents, and polymer-supported synthesis. Water soluble reagents and byproducts derived therefrom have the advantage of being easily removed by partitioning the crude reaction mixture between water (which dissolves the reagent and associated byproducts) and an organic solvent (which dissolves the desired product). Separation of the organic layer gives a purified form of the product relative to the crude reaction mixture. An example of a water soluble reagent is N-ethyl-N'-dimethylaminopropylcarbodiimide (EDC). EDC is reagent that is used in the coupling of carboxylic acids and amines to form amide bonds. EDC and the corresponding urea produced during the course of the reaction (N-ethyl-N'-dimethylaminopropylurea) are both soluble in water at low pH and can thus be washed away into an acidic water layer. The use of EDC greatly simplifies purification of the amide product relative to other carbodiimides such as N,N'-dicyclohexylcarbodiimide (DCC) which are not water soluble. Polymer-supported reagents and byproducts derived therefrom are likewise easily separated by filtration of the polymeric materials from a crude reaction mixture. An example of a polymer-supported reagent is poly(styrene-divinylbenzene)-supported triphenylphosphine which may be used in Wittig olefination and Mitsunobu reactions. The byproduct of this transformation, polymer-supported triphenylphosphine oxide, is easily removed by filtration which simplifies purification greatly compared to the solution phase reagent. The use of triphenylphosphine in solution phase Wittig reactions gives triphenylphosphine oxide as a byproduct which is difficult to completely remove except by time consuming chromatography or repeated crystallization. Polymer-supported synthesis minimizes time spent on purifications by attaching a starting material to a polymer. Subsequent synthetic transformations are carried out in such a manner that desired reactions are driven to completion on the polymer-supported material and excess reagents and byproducts in solution are subsequently removed by filtering the polymer and rinsing with solvent(s). At the end of the synthesis, the desired product is chemically cleaved from the polymer. The resulting product is typically obtained in greater purity than would be possible if all of the steps were carried out in solution with no chromatography or crystallization of synthetic intermediates. Purification in a multistep synthesis is thus largely reduced to a number of filtrations, although a single purification of the final product by conventional means is often necessary to remove byproducts resulting from the resin cleavage step. Thus, water soluble reagents, polymer-supported reagents, and polymer-supported synthesis each-provide increased efficiency reducing purification to mechanically simple liquid-liquid and liquid-solid separation methods which are easy to automate.
The increased simplicity and efficiency which allow automation of organic synthesis using the methods described above comes at the price of increased reagent cost and/or substantial synthesis development time. Water soluble reagents and polymer-supported reagents must be customized for each type of synthetic transformation. The time necessary to optimize a particular reagent significantly increases its cost. Consequently, EDC is more expensive than DCC and polystyrene-supported triphenylphosphine is more expensive than triphenylphosphine. Polymer-supported syntheses traditionally require longer development time than solution phase due to the limitations imposed by the method. One must choose the optimum polymer, develop a linking strategy which can be reversed at the end of the synthesis and find successful conditions for each reaction without many of the conventional spectral and chromatographic analysis tools that are available to solution phase synthesis. Thus, at the current state of the art, much of the time/cost saved by increasing the efficiency of purifications via the above methods is lost to increased reagent costs and/or synthetic development time.
Polymer-supported reagents have been extensively reviewed in the literature. The following citation is representative of the current state of this art: Sherrington D. C., Chem. Ind., (London) 1991;1:15-19.
Solid-supported synthesis has been extensively reviewed in the literature. The following two citations are representative of the current state of this art: Fruchtel J. S. and Jung G., Angew. Chem. Int. Ed. Engl. 1996;35:17-42, Thompson L. A. and Ellman J. A., Chem. Rev., 1996;96:555-600.
A purification process known as covalent chromatography has been described in the scientific literature. Using covalent chromatography a desired material is isolated from a complex mixture by selective reaction with a polymeric reagent, followed by filtration, and rinsing. The desired material is then liberated from the polymer by a chemical cleavage. Typically this process is applied to proteins and other macromolecules as a way of isolating them from complex mixtures of cellular components. This technique has also been applied in the separation of low molecular weight allergens from plant oils as described by Cheminat A., et al., in Tetr. Lett., 1990;617-619. Covalent chromatography differs from the instant invention in that the polymeric materials used must be both capable of covalently reacting with a desired material in a solution containing impurities and capable of subsequent cleavage of said covalent bond during the retrieval of the desired material. Polymer-supported quench methods of the present invention rely on chemically robust and ideally irreversible attachment of undesired materials that are found in the crude product of an organic reaction to a polymeric support, leaving the desired product in solution.
Polymeric reagents have been employed during the course of a reaction to enhance yield of the desired product by minimizing side reactions as described by Rubenstein M. and Patchornik A., Tetr. Lett., 1975;1445-8, but this use of a polymeric reagent does not eliminate the need for conventional purification of the desired product.
Polymeric reagents which selectively remove metal ions from solutions by chelation have been described but this use of a polymeric reagent in purification does not involve formation of covalent bonds. For a review of the current state of this art see Alexandratos S. D. and Crick D. W., Ind. Ens. Chem. Res., 1996;35:635-44.
The synthesis of dendritic polyamides on polymeric supports has been described by Ulrich K. E., et al., Polymer Bul., 1991;25:551-8. As synthetic intermediates of the synthesis, polymer-supported dendritic polyamines are described which, by virtue of the fact that they contain an easily cleaved linker, are structurally distinct from those of the present invention which contain chemically robust linkers.
Solution-phase parallel synthesis is an excellent way to form large libraries of small molecules. This is a logical extension of solid phase organic synthesis (SPOS) which has a few limitations in terms of selection of resins and an appropriate handle to hook up the resin on the substrate (Thompson L. A. and Ellman J. A., Chem. Rev., 1996;96:555; Hemikens P. H. H., Ottenheijm H. C. J., and Rees D., Tetrahedron, 1996;52:4527). Additionally, SPOS may not be compatible to a variety of reagent types and in future will need other complementary solution phase methods to give pure compounds in multi-gram quantities in good yields. Earlier, a few reports have appeared where solid phase quenchers in the form of reagents on the solid-phase (Kaldor S. W., Siegel M. G., Frita J. E., Dressman B. A., and Hahn P. J., Tetrahedron Lett., 1996;37:7193) or ion exchange resins (Gayo L. M. and Suto M. J., Tetrahedron Let., 1997;38:513) have been used for quenching reactions to eliminate the reactive components in the reaction. Boger and his coworkers (Cheng S., Comer D. D., Williams J. P., Myers P. L., and Boger D. L., J. Am. Chem. Soc., 1996;1 18:2567) have also reported an excellent protocol for multiple step solution phase parallel synthesis to synthesize final compounds in good purity and quantities.
The aforementioned references do not describe or suggest the quench reagents disclosed herein, nor do they teach the purification utility of a quench reagent in the practice of organic synthesis, of automated organic synthesis and combinatorial chemistry as described in the present invention.
Thus, we have surprisingly and unexpectedly found that one or more reagents can be added at the conclusion of an organic reaction to covalently react with excess reagents and/or unwanted byproducts. The impurities are then easily removed by conventional separation techniques leaving a solution of the desired synthetic intermediate or product which is enhanced in purity relative to the crude reaction mixture. Purification by quench is mechanically simple and rapid compared to conventional means of purification such as column chromatography, distillation or crystallization. This means of purification is readily applied to large variety of organic reactions and is amenable to both manual and automated organic synthesis environments. Hence, it is of tremendous value in the preparation of large libraries of organic molecules by automated parallel synthesis and by automated or manual combinatorial synthesis.